Caseins are the principal milk proteins and are normally synthesized and secreted only in the mammary gland during lactation. The first detailed characterization of the casein genes was done is the inventor's laboratory. Yu-Lee, et al., Nuc. Acids Res., 14:1833-1902 (1986).
Since its introduction, microinjection of DNA into the pronucleus of a fertilized one-cell embryo has been used to transfer a large number of genes into the mouse genome. Gordon et al. Proc. Natl. Acad. Sci. USA 77:7380-7384 (1980); Palmiter and Brinster, Cell 41:343-345 (1985) and Palmiter and Brinster Ann. Rev. Genet. 20 465-499 (1986). The technique is useful for studies of the specific nucleotide sequences involved in gene expression and regulation, and for its practical applications for improvement of domestic livestock. Transgenic sheep and pigs have now been produced. Hammer et al., Nature (London) 315:313-345 (1985). Studies in cattle are in progress. Kraemer et al., In: Gene Transfer in Cattle and Sheep, Banbury Report No. 20 pp. 221-227 (1985).
To produce transgenic animals of practical use in agriculture, the foreign gene must be integrated into the genome of the host animal and transmitted to its offspring; it must be expressed in the appropriate tissue; and its expression must be at a high rate and subject to normal or artificial regulatory mechanisms. Tissue specificity of transgene expression has been reported for several genes including the rat elastase I gene, Ig light and heavy chain genes, the rat myosin light chain gene and mouse/human .beta.-globin gene; Swift et al., Cell 38:639-646 (1984); Storb et al., Nature (London) 310:238-241 (1984), Grosscheldl et al., Cell 41:885-897 (1984); Shani, Nature (London) 314:283-286 (1985); and Chada, et al., Nature (London) 314:377-380 (1985). The factors directing tissue-specific expression are not fully understood. The evidence from the work with the MMTV promoter and the mouse metallothionein promoter suggests that DNA sequences in 5'-flanking DNA are important. Stewart et al., Nucl. Acids Res. 12:3895-3906 (1984) and Palmiter and Brinster, Cell 41:343-345 (1985).
Clues to this problem are beginning to emerge from studies both in transgenic animals and in cell culture systems. It is apparent that specific enhancer sequences in 5- flanking DNA, sometimes located far upstream from the transcription start site, and sequences in or close to the promoter itself, are involved in tissue-specific gene expression. Gene expression in transgenic mice has been targeted to the appropriate tissue by inclusion of 5'-flanking and/or 3'-flanking DNA from the homologous gene in the case of .beta.-globin, elastase, .alpha.-fetoprotein, .alpha.-A-crystalline and insulin. Magram et al., Nature (London) 315:338-340 (1985); Ornitz et al., Nature (London) 313:600-602 (1985); Krumlauf et al., Mol. Cell. Biol. 5:1639-1648 (1985); Overbeek et al., Proc. Natl. Acad. Sci. USA 82:7815-7819 (1985); and Hanahan, Nature (London) 315:115-121 (1985).
The insulin gene has been analyzed the most extensively. The rat insulin I gene requires both an enhancer region between --103 and -133 and the promoter region itself for expression of a marker gene in hamster insulinoma (HIT) cells compared to BHK cells. Edlund et al., Science 230:912-916, (1985). Furthermore, the rat insulin II gene requires a 530 bp 5'-flanking sequence to direct the expression of an SV40 oncogene to the .beta. cells of the pancreas in transgenic mice. Hanahan, Nature (London) 315:115-121 (1985).
The bacterial chloramphenicol acetyltransferase (CAT) gene expression has been targeted to the eye lenses by linking a -364 to +45 DNA fragment of the murine .alpha.-A-crystalline to the coding sequence of the CAT gene. Overbeek et al., Proc. Natl. Acad. Sci. USA 82; 7815-7819 (1985).
The ability to target specific genes to the mammary gland should result in the efficient synthesis and secretion of proteins, ultimately impacting the fields of biotechnology, pharmacology, medicine, food science and cancer research. For example, while a variety of expression vectors have been developed for the efficient synthesis of proteins in bacteria and yeast, in many cases the biological activity of these proteins is impaired because of the failure to correctly process these proteins. Development of mammalian cell culture systems provides an alternative strategy but the cost of these cell cultures may be prohibitive. The mammary gland provides a highly efficient in vivo model for the synthesis and secretion of grams of protein per day. The secretion continues during the lactation cycles of a mammals' life. In addition, the mammary gland contains the necessary post-translational modification systems required for the clevage, phosphorylation and glycosylation of proteins. Therefore, using this approach should make it possible to efficiently synthesize and secrete biologically important molecules. For example, proteins, hormones, growth factors, drugs, lipids and carbohydrates can be synthesized and secreted, providing new tools in medicine and pharmacology. This methodology also provides a method to manipulate the composition of mammary fluid (milk) by altering its protein, carbohydrate and lipid composition and by the inclusion of bacteriostatic agents. These changes will represent important changes in agricultural and food technology science. Additionally, the ability to target oncogenes to the mammary gland will facilitate basic breast cancer research, because it provides a model to analyze the basic mechanisms of transformation in mammary epithelial cells. This investigational methodology is not available when using in vitro cell culture systems.
The present invention provides a method that not only targets the expression of genes in the mammary gland but also provides for efficiently secreting these proteins during lactation.